Hydro-alcoholic extract of Matricaria recutita exhibited dual anti-spasmodic effect via modulation of Ca2+ channels, NO and PKA2-kinase pathway in rabbit jejunum.

OBJECTIVE
Several studies have shown the antispasmodic activity of Matricariarecutita without detailing the underlying mechanism(s). The present study was designed to determine whether the antispasmodic mechanisms of M. recutita extract mediated via histaminergic/cholinergic receptors, Ca2+channels, activation of PKA2 and NO release in isolated rabbit jejunum.


MATERIALS AND METHODS
The concentration- dependent (3 × 10-3-1.3 × 10-2 mg/ml) antispasmodic effect of the hydro-alcoholic extract of M. recutita flowers was studied in isolated rabbit jejunum. The isolated jejunum preparations were divided into seven groups, including the pharmacological probes that modulate cholinergic, histaminergic, and nitrergic receptors, as well as PKA2.


RESULTS
M. recutita inhibited spontaneous smooth muscle contractility of the jejunum in a concentration-dependent manner (3 × 10-3-1.3 × 10-2 mg/ml) and reduced both K+- and Ca2+-induced contractions, which is similar to the effect of verapamil. The antispasmodic effect of M. recutita was inhibited by H89 (a PKA2 inhibitor). The myorelaxant effect of M. recutita increased in the presence of ACh/His and H89.


CONCLUSION
M. recutita evoked antispasmodic and spasmolytic effects mediated through different signaling pathways. Our results have shown this dual inhibitory effect is mediated by blocking Ca2+ channels, activating His and ACh receptors, releasing NO, and activating PKA2.


Introduction
Matricaria recutita (German chamomile, Asteraceae Matricaria chamomilla L. var. recutita) is one of the most abundant annual medicinal herbs in the world. It has been used as an antiinflammatory drug, an analgesic for gastrointestinal pain, as well as an antiseptic, an anti-microbial, and a sedative remedy in traditional medicine in Europe and Asia (McKay, et al., 2006). Clinical and animal studies have shown that M. recutita extract is effective as a smooth muscle relaxant (Ammon, et al., 2006;mazandarani, et al., 2011). It contains potentially active chemical constituents such as terpenoids, flavonoids, coumarins, and spiroethers. However, chamomile's main active constituents are chamazulene, apigenin and bisabolol. It seems that flavonoids are the most potent antispasmodic products of M. recutita, while its other important components such as bisabolol, chamazulene, and αbisabolol have anti-inflammatory effects (Abad, et al., 2011).
Flavonoids are not produced by the human body and, generally, daily diet is the main available source for them. Different disciplines such as chemistry, biochemistry and pharmacy have provided evidence of the vital role of flavonoids (Peng, et al., 2003). Forster and Achterrath-Tuckermann studied the antispasmodic effects of M. recutita and its flavonoids in guinea-pig ileum. They found that the antispasmodic effect of M. recutita is 91% greater than that of papaverine, a myorelaxant alkaloid from the poppy plant (Achterrath-Tuckermann, et al., 1980;Forster, et al., 1980). Apigenin is a major flavonoid found in M. recutita and it is recognized for its antiphlogistic, antispasmodic and antibacterial effects (Guenwaldr, et al., 1998;Murti, et al., 2012). In terms of gastrointestinal effects, flavonoids decrease smooth muscle contractions, possibly by interfering with calcium movement through cell membranes (Di Carlo, et al., 1999).
Among numerous studies on the antispasmodic mechanism of M. recutita, few have considered downstream signaling cascade of this plant, especially NO and PKA 2 pathways. In the present study, we hypothesized that M. recutita induces its anti-spasmodic effect via several signaling cascades including Ca 2+ channel blockade, NO release, ACh/His receptors, and PKA 2 activation. Here, we investigated 1) the concentration-dependent anti-spasmodic effect of M. recutita in the presence of contractions induced by K + and Ca 2+ , 2) anti-spasmodic mechanism of M. recutita in the presence of the ACh/His blocker, and NOS and PKA 2 inhibitors.

Drugs
Acetylcholine chloride (ACh), atropine sulfate, histamine dihydrochloride (His), cetirizine, L-NAME, l-arginine, potassium chloride, calcium chloride, verapamil hydrochloride, and H-89 were obtained from Sigma Chemicals Company, St. Louis, USA. All drugs were dissolved in distilled water and diluted in fresh Tyrode's solution.
The hydro-alcoholic extract of M. recutita was obtained from Niak Pharmaceutical Company, Gorgan, Iran.

Plant material and phytochemical analysis of extract
All phytochemical procedures were performed as previously described (mazandarani et al., 2011). Fresh M. recutita flowers were harvested from a farm by Niak Pharmaceutical Company (Golestan province, Iran) in May 2014. The plant was identified in the herbarium of Azad University, Gorgan, Iran (RCMP-0100 code). M. recutita flowers were immediately separated, dried in an oven at 40 ºC, milled to a coarse powder (Mesh. 100) and extracted by the maceration method over 6 days using ethanol 70%. This hydro-alcoholic extract was then filtered and dried in vacuum evaporator (at 40-50 ºC). The percentage dried weight of the extract was 30.80% (Mohammedi, et al., 2011).
To analyze the phenolic and flavonoid contents of the extract, a 2.5 g sample of the dried extract was used. For each treatment, analyses were performed in triplicate.

Determination of total phenolic and flavonoid content
The total phenolic content of the extract was determined by the Folin-Ciocalteu method. Briefly, 0.2 ml of the extract was mixed with 0.5 ml of Folin-Ciocalteu reagent, 1.5 ml of 20% Na 2 CO 3 (Merck, Germany) solution, and 7.8 ml of distilled water. The sample was left for 2 hr at room temperature and its absorbance at 765 nm was determined using a UV-visible spectrophotometer (Shimadzu, Japan). The result was expressed in terms of gallic acid (Merck, Germany) equivalent mg/g dry weight of the extract. The amount of flavonoids was determined with the colorimetric method by using aluminum chloride hexahydrate (AlCl 3 .6H 2 O; Merck, Germany). Absorbance at 395 nm was read using a UV-vis spectrophotometer (Shimadzu 1601). The result was expressed in terms of apigenin (Merck, Germany) equivalent mg/g dry weight of the extract. The total phenolic and flavonoid contents of the extract were 17.2 ± 1.3% and 5.6 ± 0.4%, respectively.

Animals
In this study, we used male New Zealand white rabbits weighing 1.8-2.5 kg (Pasteur Institute of Iran, Iran). These rabbits were housed in the animal house of Golestan University of Medical Sciences, Iran, under standard laboratory conditions and provided with animal food pellets and free access to water. However, food supply was ceased 12 hr prior to experimentation. The study was approved by the Institutional Animal Ethics Committee and carried out according to Golestan University of Medical Sciences' guidelines for the use and care of laboratory animals.

Isolated tissue preparations
The anti-spasmodic activities of M. recutita were studied using isolated tissue from rabbit jejunum. The experiments were performed using jejunum sections taken from five rabbits. The rabbits were anaesthetized with sodium pentobarbital (40 mg/kg), and heparin (200 IU/kg) was used as an anticoagulant. The abdomen was exposed and opened. Several pieces of 2cm-long of jejunum were taken, cleaned, and transferred into a Petri dish containing Tyrode's solution, where it was detached from the mesenteric attachments. Each jejunum segment was mounted vertically (with open lumen) under a resting tension of 0-20 g in 25 mL tissue baths containing Tyrode's solution, connected to an isometric force transducer (N.TRI 202P, AD Instruments Pty Ltd., Sydney, Australia), and allowed to equilibrate for 20 min. The tissue-bathing medium was maintained at 37 °C (pH 7.4), and gassed with 95% oxygen and 5% carbon dioxide. The composition (mM) of the Tyrode's solution used herein was as follows: NaCl (136.9 mM), KCl (2.7 mM), CaCl 2 (1.8 mM), MgCl 2 (1 mM), NaHCO 3 (11.9 mM), NaH2PO 4 (0.42 mM), and glucose (5.5 mM). For determination of Ca 2+ channel blocking (CCB) activity, K + (50 mM) was used to induce stable contraction of the preparations, as previously described (Farre, et al., 1991). When stable and sustained contraction was achieved, verapamil was added cumulatively to obtain the concentration-dependent inhibitory response (Van Rossum, 1963). To confirm the Ca 2+ antagonist activity of the extract, the tissue was allowed to stabilize in normal Tyrode's solution, which was then washed out with Ca 2+ -free Tyrode's solution containing EDTA (0.1 mM) for 20 min. This solution was then replaced with K + -rich and Ca 2+ -free Tyrode's solution of the following composition: KCl 50 mM, NaCl 91.04 mM, MgCl 2 1.05 mM, NaHCO 3 11.90 mM, NaH 2 PO 4 0.42 mM, glucose 5.55 mM, and EDTA 0.1 mM. Following incubation for 20 min, concentrationresponse curves (CRCs) control of Ca 2+ was obtained. When the CRCs of Ca 2+ were found to be superimposable (usually after two cycles), the tissue was pretreated with the extract for 30 min to test the CCB effect. The CRCs of Ca 2+ were reconstructed at different concentrations of M. recutita and other drugs.

Statistical analysis
All results are presented as mean ± SEM. All data were tested for normality before applying the statistical tests. Two groups of experimental data were compared using Student's t-test. Concentration-dependent changes were examined using repeated-measures analysis of variance (ANOVA) and Scheffe contrasts. Changes in force were presented as a percentage of the maximal response induced by agonists (Acetylcholine, Histamine, KCL, and Ca 2+ ). The EC 50 values were obtained from cumulative concentration-effect curves via non-linear regression analysis of data points from individual concentration-effect curves plotted using GraphPad Prism 5 (GraphPad Software, Inc., CA, USA). A probability of 5% or lower was considered statistically significant.
Histamine ( L-arginine (100 mM) significantly reduced the antispasmodic effect of M. recutita. The inhibitory effect of M. recutita (3 × 10 -3 -13 × 10 -3 mg/ml) on the intestinal smooth muscle was abrogated in the presence of L-name and L-arginine (EC 50 = 1.99 × 10 -8 mg/ml and 388.9 mg/ml), respectively (n = 5). (Figures 4c  and d and Figures 5c and d) In particular, to determine the possible involvement of PKA 2 and NO in the anticontractility effects of M. recutita, we used H89, a specific inhibitor of PKA 2 . Applying H89 (1 µM) in the presence of M. recutita increased tissue relaxation by about 53%. Furthermore, addition of histamine increased the inhibitory activity of the extract by about 38%, whereas ACh and L-arginine reduced its antispasmodic activity by 16% and 10%, respectively (Figures 4c and d and Figures 5c and d).

Discussion
In the present study, we demonstrated the anti-spasmodic and spasmolytic (relaxant) effects of the flavonoid components of M. recutita (3 × 10 -3 -1.3×10 -2 mg/ml) in isolated rabbit jejunum. M. recutita evoked a spasmolytic effect on K + -induced spontaneous contractions, suggesting that calcium channels are involved in its spasmolytic actions. This effect was abrogated by L-NAME and amplified by H89. Consistent with this, the spasmolytic effect of M. recutita was mediated through the NO/PKA 2 pathway. Our study showed that the spasmolytic effect of M. recutita was mediated through NO-activated cholinergic and histaminergic receptors.
In the present study, M. recutita prevented contractions induced by high potassium concentration, suggesting the possible role of voltage-dependent Ca 2+ channels in its mechanism of action. It is well-documented that KCl induces smooth muscle contractions by activating calcium channels (Cortes, et al., 2006). Therefore, this data indicated the indirect Ca 2+ channel blocking activity of M. recutita and its flavonoid fraction. Consistent with this, several studies have shown the potential Ca 2+ channel blocking activity of M. recutita extract (Achterrath-Tuckermann et al., 1980;Forster et al., 1980;Rotondo, et al., 2009). In contrast, verapamil did not prevent the antispasmodic activity of M. recutita, suggesting that the M. recutita extract has a wide range of biological activity. M. recutita at low doses could augment the effects of verapamil and this is irrespective to indirect reaction with Ca 2+ channels.
Moreover, atropine and cetirizine blocked the spasmolytic effect of M. recutita, which probably shows a direct receptor-mediated effect of M. recutita. This possibly indicates direct mediatory roles of ACh/His receptors in the mechanism of action of M. recutita. In addition, several studies have proved the direct role of ACh and His in the muscle relaxant activity of M. recutita . Taken together, M. recutita-induced muscle relaxation, was partly mediated through ACh and His receptors.
Our data have shown that an NOS inhibitor (L-NAME) abrogated the relaxing effect of M. recutita. Along the lines of our results, Achterrath-Tuckermann et al. reported that M. recutita induced a greater anti-contractility effect in the presence of NO donors (Achterrath-Tuckermann et al., 1980). We hypothesized that the muscle relaxant effect of M. recutita is mediated by the release NO and activation of ACh/His receptors.
Indeed, a study has demonstrated that ACh/His-induced smooth muscle contraction is mediated by a NO-dependent pathway (Grifoni, et al., 2000). Taken together, in the present study, M. recutita-induced smooth muscle relaxant activity was mediated via NO release and ACh/His receptors. Precise muscle relaxant signaling mechanism of M. recutita is yet to be determined.
Furthermore, our findings showed that the inhibitory effect of M. recutita on jejunum contraction is abrogated by Larginine, due to an unknown mechanism. Applying of high concentrations of Larginine can induce acidosis and this indirectly prevents the M. recutita action. In a separate group of the experiment, we adjusted the pH of medium and interestingly L-arginine showed the previous profile of action. Consistent with this data, few studies have shown that the inhibitory effect of M. recutita on the contractile tone of smooth muscle was restored by L-arginine, indicating a controversial dual role of NO as an inhibitor or a stimulator (Michel, et al., 2011;Westfall, et al., 2011). Consistent with this, it can be concluded that the controversial dual role of NO in the signaling cascades of M. recutita on intestinal motility depends on the basal contractility state of smooth muscle, and it is observable as a spasmolytic effect on contracting smooth muscle and a tonicising effect on relaxed muscle. Ammon et al indicated the dual activity of STW5 in both hypotonic and spastic states of isolated guinea pig ileum (Ammon et al., 2006). Taken together, NO and ACh/His receptors involved in the signaling cascades of M. recutita intestinal smooth muscle contractility A previous study demonstrated that NO-evoked smooth muscle relaxation was induced by increasing cGMP and PKA 2 (Hobbs, et al., 1996). To determine the possible role of PKA 2 modulation in NOinduced smooth muscle relaxation, we analyzed the dual anti-spasmodic effects of M. recutita in the presence of a selective PKA 2 inhibitor.
Our results showed that the effects of M. recutita were mediated directly or indirectly via PKA 2 . In fact, the inhibitory effects of M. recutita on jejunum contractions were augmented by H-89, indicating that the inhibitory effect of M. recutita was mediated through PKA 2 activation. In agreement with our data, a study has shown that the spasmolytic effect of M. recutita is mediated by the inhibition of cAMP phosphodiesterase (Maschi, et al., 2008). In addition, the muscle relaxant function of GI smooth muscle tone was regulated by cAMP-PDE pathway and the flavonoid fraction of M. recutita inhibits cAMP activity (Murthy, 2006). Therefore, our data indicated that the PKA 2 -cAMP transduction pathway was involved in the inhibitory effect of M. recutita on smooth muscle contraction.
Consistent with our results, it has been proven that the gastrointestinal (GI) smooth muscles are synchronized by different mechanisms including the histaminergic, cholinergic, nitrergic pathways to stimulate PKA 2 signaling cascade (Kochar, et al., 2011;Murthy, 2006). Moreover, PKA acts as a downstream and upstream signaling cascade to induce relaxation via several mechanisms, in smooth muscle cells.
As noted above, PKA and PKG act on upstream targets to regulate cAMP and cGMP levels by desensitizing receptors and stimulating PDE activities (Murthy, 2006). Moreover, a study demonstrated that the activation of ACh and His receptors is abolished by PKC inhibitors (Eto, et al., 2001).
In line with the aforementioned data, in our study, H89 potentiated smooth muscle relaxation induced by M. recutita, and this effect was decreased in the presence of ACh and L-arginine.
Therefore, we suggest that specific interplay among different signaling cascades including PKA 2 , ACh, His, and adrenergic receptors, as well as the nitrergic pathway, is required to mediate the antispasmodic effect of M. recutita in rabbit intestinal smooth muscle.
In this study, we were not able to determine the sequential pathway of NO and PKA 2 activation in mediating the antispasmodic effects of M. recutita following the stimulation of ACh/His receptors. However, future studies should be conducted to provide sufficient evidence related to the downstream signaling pathways of M. recutita and focus on its possible link to ACh/Hisreceptors and the NO pathway.
In the present study, the concentrationresponse curves of M. recutita and its pure flavonoid fraction exhibited similar spasmolytic activity without significant changes in the EC 50 values. Hence, the fundamental role of flavonoids in the antispasmodic effects of M. recutita was confirmed. Indeed, the total flavonoid fraction of M. recutita is used to prevent interfering actions of essential oils. Our previous study showed high amounts of apigenin and quercetin in the hydroalcoholic extract of M. recutita (mazandarani et al., 2011). Thus, we suggest that the flavonoid fraction of M. recutita actively induces anti-spasmodic action. Moreover, a growing body of evidence suggests that apigenin and quercetin exhibit smooth muscle antispasmodic activities through a wide range of signaling cascades (Amira, et al., 2008;Gharzouli, et al., 2004). Consistent with our results, the myorelaxant activity of apigenin has been attributed to the inhibition of Ca 2+ release or induction of eNOS and NO production in mouse gastric tissue (Chen, et al., 2010). In addition, Rotondo et al., showed significant NO production in response to an increase in intracellular calcium concentration by apigenin-and quercetin-induced gastric relaxation (Rotondo et al., 2009). Thus, we suggest that the total flavonoid fraction of M. recutita (apigenin, quercetin, etc.) mediates its inhibitory effect on smooth muscle contractions through blockade of calcium release and NO production.
The myorelaxant activity M. recutita is observed as dual anti-spasmodic and spasmolytic effects. Considering the fundamental role of Ca 2+ channels as a possible end-effector step in the mechanism of action of M. recutita, we suggest that NO, His/ACh/ adrenergic receptors, and PKA 2 are the main signaling components of M. recutita in the presence of spasmodic agents. Nevertheless, without analyzing the influence of M. recutita in the presence of several inhibitors or detecting direct kinase activity assay, we cannot determine whether the interactions among multiple mediators take place in parallel or in sequence. Meanwhile, further study is required to elucidate the mechanism of action of the various flavonoid components of M. recutita and the contributions of various signaling processes in the curative properties of this plant when uses in the treatment of gastrointestinal motility disorders. Acknowledgment